The ability to determine DNA sequences is crucial for understanding the function and control of genes and for applying many of the basic techniques of molecular biology. Native DNA consists of two linear polymers, or strands of nucleotides. Each strand is a chain of nucleosides linked by phosphotidester bonds. The two strands are held together in an antiparallel orientation by hydrogen bonds between complementary bases of the nucleotides of the two strands: deoxyandenosine (A) pairs with thymidine (T) and deoxyguanosine (G) pairs with deoxycytidine (C).
Presently there are two basic approaches to DNA sequence determination: the dideoxy chain termination method, e.g. Sanger et al, Proc. Natl. Acd. Sci., Vol. 74, pgs, 5463-5467 (1977); and the chemical degradation method, e.g. Maxam et al, Proc. Natl. Acad. Sci., Vol. 74, pgs. 560-564 (1977). The chain termination method has been improved in several ways, and serves as the basis for all currently available automated DNA sequencing machines, e.g. Sanger et al, J. Mol. Biol., Vol. 143, pgs, 161-178 (1980); Schreier et al. J. Mol. Biol., Vol. 129, pgs, 169-172 (1979); Mills et al, Proc. Natl. Acad. Sci., Vol. 76, pgs, 2232-2235 (1979); Smith et al, Nucleic Acids Research, Vol. 13, pgs, 2399-2412 (1985); Smith et al., Nature, Vol. 321, pgs, 674-679 (1987); Prober et al, Science, Vol. 238, pgs, 336-341 (1987), Section II, Meth. Enzymol., Vol. 155, pgs, 51-334 (1987); Church et al, Science, Vol. 240, pgs, 185-188 (1988); Tabor et al, Proc. Natl. Acad. Sci., Vol. 84, pgs, 4767-4771 (1987); Tabor et al, Proc. Natl. Acad. Sci., Vol. 86, pgs, 4076-4080 (1989); Innis et al., Proc. Natl. Acad. Sci., Vol. 85, pgs, 9436-9440 (1988); and Connell et al., Biotechniques, Vol. 5, pgs, 342-348 (1987).
Both the chain termination and chemical degradation methods require the generation of one or more sets of labeled DNA fragments, each having a common origin and each terminating with a known base. The set or sets of fragments must then be separated by size to obtain sequence information. In both methods, the DNA fragments are separated by high resolution gel electrophoresis. Usually, the DNA fragments are labelled by way of radioactive nucleoside triphosphate precursors or by way of fluorescent dyes.
In most automated DNA sequencing machines, fragments having different terminating bases are labeled with different fluorescent dyes, which are attached either to a primer, e.g. Smith et al (1987, cited above), or to the base of a terminal dideoxynucleotide, e.g. Prober et al (cited above). The fluorescently labeled fragments are combined and loaded onto the same gel column for electrophoretic separation. Base sequence is determined by analyzing the fluorescent signals emitted by the fragments as they pass a stationary detector during the separation process.
Such analysis has been complicated by a number of phenomena, such as band compression, overlapping emission bands of the fluorescent labels, the appearance of artifactual bands, noise due to misincorporation of labeled dideoxynucleosides, and bands that vary widely in intensity in a highly sequence dependent manner, e.g. Mills et al (cited above), Connell et al (cited above), and Tabor et al (1987 and 1989, cited above). The latter two phenomena are believed to be caused by difficulties that some DNA polymerases have in reading through certain template sequences and/or in accommodating some commonly used nucleoside analogs. Band uniformity has been improved in many instances by the use of a modified T7 DNA polymerase (Sequenase.TM.) and the substitution of Mn.sup.+2 for MG.sup.+2 as the divalent cation in the polymerase reactions, Tabor et al (1987 and 1989, cited above). However, the problem persists and still presents an important limitation to current automated DNA sequencing approaches.